The main goal of this proposal is to examine the role and significance of altered expression of homeotic genes, caused by DNA methylation changes, in the pathogenesis and progression of breast cancer. Changes in DNA methylation are frequently detectable epigenetic abnormalities in human neoplasia. We performed an unbiased evaluation of DNA methylation alterations over an entire genome using a technique named Methylation Sensitive-Amplified Fragment Length Polymorphism (MS-AFLP). In that study, we analyzed normal and tumor tissue MS-AFLP methylation fingerprints of breast, prostate, and colon cancer patients. We found that several of the fingerprint bands exhibiting tumor specific intensity changes represented DNA fragments from diverse homeotic gene expression. In concert with the unbiased nature of the MS-AFLP fingerprinting approach, we believe that our observation is the tip of the iceberg, and that DNA methylation changes in homeotic genes might be extremely frequent events in carcinogenesis. Homeoproteins are transcription factors that direct embryogenesis and cell differentiation. Therefore, epigenetic methylation alterations of homeotic genes may trigger a cascade of changes in expression of many genes, resulting in a cell with a less differentiated and less positionally restricted phenotype if those methylation alterations are accompanied by the alterations in homeotic gene expression. We believe that both DNA methylation and homeotic genes are important in breast carcinogenesis. We will test this hypothesis by proposing two specific aims: First, we will analyze DNA methylation and gene expression alterations in various homeotic genes in breast tumor tissues and identify common breast cancer-specific alterations. Cancer-specific alterations and cell type- specific alterations will be discriminated by analyzing the changes in microscopically selected uniform populations of normal and cancer cells. We will next examine the functionality of altered expression of homeotic genes in breast carcinogenesis. Expression constructs will be prepared for those homeotic genes that are hypermethylated and down-regulated in expression and introduced into non-expressor breast cancer cells. Effects of the homeotic gene expression on the expression of other genes as well as on cellular phenotypes (anchorage dependent/independent growth and motility) will be investigated. Both the constitutive and inducible expression systems will be used to examine the tumor suppressor function of these homeotic genes.